Androgen depletion, which blocks the production or action of androgens, serves as an effective treatment for locally advanced or metastatic prostate cancer (PCa). The main limitation of androgen depletion is that PCa frequently recurs with an aggressive and lethal androgen depletion-independent (ADI) phenotype. Clinical and experimental studies of PCa recurrence during androgen depletion have revealed that although ADI PCa may no longer be fully dependent on androgens, activity of the androgen receptor (AR) remains critical for continued growth. Therefore, the AR is regarded as a viable target for treating ADI PCa. We have been studying the mechanisms of AR activation in models of PCa progression. The AR is able to achieve activity in the absence of androgens in ADI, but not androgen dependent PCa cells. However, unlike ligand-induced AR activation, ligand-independent AR activation does not require the AR ligand binding/activation function (AF)-2 module in the AR C-terminal domain (CTD), which is the ultimate target of androgen depletion. Rather, ligand- independent AR activity requires transactivation unit (TAU)-5 in the poorly-characterized AR N-terminal domain (NTD). AR TAU5 maps to a discrete WHTLF motif, which functions as a novel transcriptional activation domain. Importantly, WHTLF activity is required for AR activation under conditions of no/low androgens, only in ADI PCa cells. A shift from AR AF-2-dependent transcriptional activation in androgen-dependent PCa cells to WHTLF-dependent transcriptional activation in ADI PCa cells provides a rational, mechanism-based explanation for PCa resistance to therapies that target the AR CTD. Such a mechanism is strongly supported by the recent discovery of novel, constitutively active AR splice isoforms that lack the entire AR CTD and can support the growth of ADI PCa cells during androgen depletion. We therefore hypothesize that androgen depletion exerts a selective pressure on PCa cells to shift AR transcriptional activation domain dependence from AF-2 to WHTLF, which allows the AR to activate target genes and support continued PCa growth. The goals of this proposal are to 1) Determine the relative roles of AR AF-2 and WHTLF for supporting the growth of ADI PCa cells;and 2) Identify the mechanisms by which the AR WHTLF motif is able to activate transcription. Overall, these studies will establish the relative contributions of discrete AR transcriptional activation domains to the growth of ADI PCa, and identify molecules that mediate transcriptional activity of the AR WHTLF motif. Ultimately, these studies will determine whether WHTLF may represent a novel functional AR domain that could be targeted to treat ADI PCa. PUBLIC HEALTH RELEVANCE: In the United States and other industrialized countries, prostate cancer is the most frequently diagnosed male cancer and second leading cause of male cancer deaths. The androgen receptor is a key regulator involved in prostate cancer development, growth, treatment, and progression to a lethal androgen depletion-independent phenotype. The goal of this proposal is to define the mechanisms by which the androgen receptor supports the growth of androgen depletion-independent prostate cancer and identify novel targets to treat this disease.